Human breast carcinoma is thought to develop through progressive stages from atypical hyperplasias to in situ carcinoma and finally to invasive and metastatic cancer. In situ breast carcinoma consists of small, isolated neoplastic foci which cannot be selectively studied by conventional methods. In this study, we used tissue microdissection to examine the loss of heterozygosity (LOH) of chromosome 11q13 in both in situ and invasive lesions of the breast, as compared to normal breast epithelium from the same patients. Forty-one cases of sporadic breast cancer were analyzed. Tissue microdissection allows for procurement and PCR-based analysis of small lesions using either frozen or formalin-fixed, paraffin-embedded tissue sections. LOH on chromosome 11q13 was found in 24 of 36 (67%) of the informative invasive breast cancer cases using two polymorphic DNA markers specific for this region (INT2 and PYGM). Twenty-one of the cases which demonstrated LOH in the invasive tumor also contained in situ carcinoma in the same tissue section. Seventy-one % (15 of 21) of the microdissected in situ lesions showed LOH of chromosome 11q13. Every case (15 of 15) of in situ tumor with LOH showed loss of the same allele in the corresponding invasive tumor cells. The results of this study suggest that a tumor suppressor gene located on chromosome 11q13 may play an important role in the early stages of development of sporadic human breast cancer. This finding provides molecular genetic support for the hypothesis that invasive breast cancer arises from in situ lesions.